Cyanide-free reagent and method for the determination of hemoglobin

ABSTRACT

A cyanide-free method and reagent for determining the concentration of total hemoglobin in a whole blood sample accurately in less than 10 seconds comprising a ligand selected from the group consisting of imidiazole, imidazole derivatives, N-hydroxyacetamide, N-hydroxyl amine, pyridine, oxazole, thiazole, pyrazole, pyrimidine, purine, quinoline, and isoquinoline, and a surfactant with strong erythrolytic capability selected from the group consisting of lauryl dimethylamine oxide and octylphenoxy polyethoxyethanol. The reagent pH is adjusted to about 11 to about 14. Rapid mixing of the reagent with a blood sample leads to the formation of a stable chromogen whose absorbance can be measured between 540 and 550 nm. The cyanide-free reagent is ideal for use on an automated high through-put clinical hematology analyzer.

This application is a continuation of application Ser. No. 08/212,626filed Mar. 11, 1994 now abandoned.

TECHNICAL HELD

The present invention relates generally to reagents and methods for usein determining the concentration of hemoglobin present in a whole bloodsample, either in manual methods or automated methods. The specificreagent rapidly converts all of the hemoglobin in the sample into a welldefined chromogen for detection and measurement, without the use ofcyanide.

BACKGROUND OF THE INVENTION

The science of hematology has long recognized the importance ofmeasuring the amount of hemoglobin in a blood sample since it is thehemoglobin molecule which transports oxygen from the lungs to thevarious tissues and organs of the body. The accurate measurement ofhemoglobin concentration in a patient's blood is arguably the mostimportant parameter determined in a hematology analysis. The hemoglobinconcentration is used to screen for anemia which in turn is a sign ofunderlying disease.

In countries with a "Western Diet", a hemoglobin concentration below 14grams per deciliter (g/dL) in men and 12 g/dL in women is indicative ofanemia. The causes of anemia are many and a low hemoglobin concentrationis a strong signal for a thorough workup by the patient's physician.Therefore an accurate and reliable method for measuring hemoglobinconcentration is a vital part of any hematology system. The two mostcommon reasons for a patient to be anemic are blood loss and dietarydeficiencies in iron, vitamin B12, or Folic Acid. In the former case itis mandatory for the physician to determine the cause of the blood lossand treat it. And again in the latter case, a proper diagnosis is neededto define the appropriate nutritional supplemental treatment.

In addition to its importance as the primary indicator of anemia, thehemoglobin concentration is used in combination with other blood cellparameters to calculate several indices. The Mean Corpuscular Hemoglobin(MCH) is the mass of hemoglobin per red cell and is calculated bydividing the hemoglobin concentration by the number of Red Blood Cellsin the comparable volume. The Mean Corpuscular Hemoglobin Concentration(MCHC) is the weight percent of hemoglobin in a Red Blood Cell and iscalculated by dividing the hemoglobin concentration by the hematocritand converting to percent. Both MCH and MCHC are useful parameters inthe diagnosis of an anemia. Their importance further emphasizes thenecessity of making an accurate and reliable measurement of thehemoglobin concentration in a blood sample.

Modern methods of measuring the hemoglobin content utilizespectrophotometry to quantitate the amount of the oxygen-carryingprotein in the sample. The requirements of any spectrophotometric methodto measure hemoglobin in a blood sample are two-fold:

1. The method must release all the hemoglobin from the red blood cell inwhich it is sequestered; and

2. The method must convert all the hemoglobin in the sample into asingle chromogenic species, regardless of what form the hemoglobin is inwhen the reaction is begun.

The first requirement can be achieved by many means; the simplest beingto dilute the blood sample in distilled water to effect a hypotoniclysis. However, modern automated hematology instruments require a morerapid lysis than can be achieved with hypotonic lysis. Frequently,surfactants are added to the lysing reagent to hasten the release ofhemoglobin and to clear any turbidity which may be in the sample due toelevated lipid content. Various kinds of surfactants are suitable forthis task, including anionic, non-ionic, zwitterionic, and cationic. Theamount of surfactant required can range from about 100 mg/L to about 50g/L, depending on the "potency" of the surfactant and the ionic strengthof the reagent.

The second requirement necessitates an understanding of the chemistry ofthe heme iron which carries oxygen when complexed in a globin proteinmolecule. The heme iron is maintained in the +2 (Fe^(II)) oxidationstate in a normal blood sample. Since the blood sample is usually takenfrom a vein, the hemoglobin is mostly in the de-oxy state; that is, nooxygen is bound to the heme iron. However, as soon as the sample comesinto contact with the atmosphere or is diluted into an oxygen containingbuffer or lysing reagent, it is rapidly converted into oxy-hemoglobin;the heme iron binds oxygen but stays in the Fe^(II) (reduced) state. Inmany cases, the amount of hemoglobin in the sample could be determinedfrom the oxy-hemoglobin chromogen which is formed naturally uponexposure to air. However, there are some conditions which make thissimple solution unacceptable. In some diseases, genetic conditions, orpoisonings, a patient may have a significant amount of met hemoglobin incirculation. In met hemoglobin, the heme iron is in the +3, (Fe^(III))oxidized state. It cannot bind oxygen, nor can it readily be reduced toFe^(II) so that it can bind oxygen to be measured as oxy hemoglobin.Also, heavy cigarette smokers and workers exposed to high concentrationsof automobile exhaust frequently accumulate a high concentration ofcarbon monoxide bound to their heme iron. Carbon monoxide is tightlybound and blocks the binding of oxygen, thereby causing an error in theconcentration of hemoglobin if determined by the oxy hemoglobin method.The most commonly used approach to the measurement of hemoglobin is tooxidize all the heme iron to the +3 state and to introduce a ligandwhich will quantitatively bind to all the heme iron to produce a singlechromogenic species for quantitation by spectrophotometry.

The classical method is that of Drabkin. Briefly, the hemoglobin isreleased by hypotonic lysis (modern adaptations have added surfactantsto speed the lysis), the heme iron is oxidized to Fe^(III) by means ofpotassium ferricyanide, and the iron reacted with the cyanide anion ofpotassium cyanide. Cyanide binds very tightly to Fe^(III) and gives adistinctive chromogen with a peak at about 540 nm. Recent adaptationshave involved the deletion of the ferricyanide oxidizing agent and havedepended on the oxidation of the heme iron by atmospheric oxygen (oroxygen equilibrated reagents) at elevated pH in the presence ofsurfactants; cyanide is still used as the heme iron ligand in mostprocedures in spite of its well-known toxicity.

Many automated hematology analyzers utilize a modification of Drabkin'smethod. In these methods, red cells are lysed by a cationic surfactantat pH above 10 in the presence of cyanide. Under these conditions, whitecell nuclei remain intact and can be counted by common impedancemethods. Hemoglobin is measured by taking the optical density of thesame solution at 540 nm, as is customarily done with Drabkin's method.The method can yield erroneous measurements in samples which have a highwhite cell count because of turbidity due to scattering of light by thenuclei. Lipemic and icteric samples also can interfere due to turbidityor increased absorbance of the sample. The present invention avoidsthese problems by providing a reagent which measures hemoglobin freefrom interference due to other blood components.

Stroupe, et al. (U.S. Pat. No. 4,200,435) disclose the use of imidazoleas a ligand for the determination of glycosylated hemoglobin in thepresence of an allosteric effector. They also disclose the use of asurfactant lysing agent, an oxidizing agent, and a heme-binding ligandto determine the amount of hemoglobin in a sample. Stroupe, et al. (U.S.Pat. No. 4,255,385) also disclose reagent kits containing the abovereagents. The present invention differs from that disclosed by theStroupe, et al. patents in that it requires no added oxidizing agent andin that the reaction is completed within 10 seconds as opposed toapproximately 10 minutes in the prior art invention.

Benezra, et al. (U.S. Pat. No. 4,853,338) disclose a cyanide-freehemoglobin reagent which comprises a very high surfactant concentration(20 to 50 g/L) and a pH between 11.3 and 13.7. From the elevated pH, andthe large amount of NaOH required to achieve the desired pH, it can beinferred that the heme-binding ligand of this invention is the hydroxideanion. If the hydroxide ion content of the reagent is not high enough,the heme will not be converted stoichiometrically to a single chromogensince water which is present at high (55M) concentration will competefor iron binding sites and may give an erroneous result. The presentinvention avoids this problem.

It is an object of the present invention to provide a cyanide-freemethod and reagent for the determination of total hemoglobin present ina whole blood sample. It is a further object of this invention toprovide a rapid method for total hemoglobin determination which can beused on automated instruments. It is another object of this invention toprovide a cost effective reagent. It is another object of this inventionto provide a method for total hemoglobin determination in whole bloodwithout the interference of other blood components.

These and further objects of the invention will become apparent to thoseof ordinary skill in the art from the following description and figures.

SUMMARY OF THE INVENTION

A cyanide-free reagent for use in a hemoglobin determination methodcomprises an aqueous solution of (i) a cyanide-free ligand selected fromthe group consisting of imidazole, imidazole derivatives,N-hydroxyacetamide, N-hydroxyl amine, pyridine, oxazole, thiazole,pyrazole, pyrimidine, purine, quinoline, and isoquinoline, and presentat a concentration of 0.1 to 2.0 Molar and (ii) a strong erythrolyticsurfactant selected from the group consisting of lauryl dimethylamineoxide and octylphenoxy ethanol, and present in an amount from about 0.1%to about 1.0% (w/v), the pH of the reagent being adjusted to 11-14,preferably with a monovalent base. According to the method of theinvention, the cyanide-free reagent is rapidly mixed with the bloodsample to form a chromogen. The absorbance, or optical density, of theresulting chromogen is then measured as an indication of theconcentration of hemoglobin. The chromogen formed in this method can beobtained in approximately ten (10) seconds, making it especially wellsuited for use in automated instruments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a spectrum of a sample prepared using the method and reagentof the present invention.

FIG. 2 is a correlation plot of hemoglobin data obtained using acyanide-containing hemoglobin method performed on a Cell-Dyn analyzervs. the cyanide-free hemoglobin method of this invention which wasperformed on an experimental, automated hematology analyzer.

FIG. 3 is a correlation plot of hemoglobin data obtained using thecyanide-containing hemoglobin method performed on a Coulter STKS vs. theinstant cyanide-free hemoglobin method performed on an experimental,automated hematology analyzer.

FIG. 4 is a correlation plot of the hemoglobin data for a freshpreparation control of the cyanide-free hemoglobin reagent of thisinvention vs. a six (6) month old preparation of the cyanide-freereagent.

FIGS. 5A and B show the kinetics of reaction for a control and thecyanide-free hemoglobin reagent of the present invention, when stored at45° C. for three (3) months.

DETAILED DESCRIPTION OF THE INVENTION

The reagent of the present invention was developed for the totalhemoglobin analysis when used on a high through-put automated hematologyinstrument.

A reagent useful for hemoglobin determination must be able to quicklylyse the erythrocytes and rapidly complex with the hemoglobin so that adetectable chromogenic structure is formed for detection andmeasurement. The present invention is particularly advantageous becausethe complex is formed rapidly and remains stable for a period of timecompatible with automated instruments. The reactant is stable for manyweeks. The present invention is also particularly advantageous becausethe resulting chromogen appears to be free of interference from otherblood components and can be measured at wavelengths in the spectralrange of automated hematology instruments already in the field. Forcomparison purposes, the cyan met hemoglobin method typically measuresabsorbance at 540 nm. A reddish brown chromogen can be formed accordingto the present invention which has an absorption maximum at about 544nm.

The reagent of the present invention is an aqueous solution of aligand-forming compound such as imidazole and imidazole derivatives. Theligand-forming compound is present at concentrations of 0.1M to 2.0M.Imidazole, from the present reagent, ligates with the hemoglobin whichis released from the erythrocytes in the sample. Other ligand-formingcompounds useful in the present invention include N-hydroxyacetamide,N-hydroxyl amine, pyridine, oxazole, thiazole, pyrazole, pyrimidine,purine, quinoline, and isoquinoline. Anions which can bind the oxidizediron heme include cyanate, fluoride, azide, nitrite, hydroxide, acetate,and formate; acceptable salts of these anions include sodium, potassium,ammonium, and the like.

The reagent further contains a surfactant with a strong erythrolyticcapability. Lauryl dimethylamine oxide (Ammonix L.O.) [Stepan ChemicalCompany, Northfield, Ill.], and octylphenoxy polyethoxyethanol(Triton×100) or other strong detergents may be used as the surfactantcomponent of the lysing reagent. The surfactant should be present atconcentrations from about 0.1% to about 1.0% (w/v). The pH of thereagent should be adjusted to between 11 and 14, preferably 12.5.Monovalent bases such as sodium hydroxide and potassium hydroxide may beutilized for pH adjustment.

According to the method of the present invention, the lysing reagent ismixed with a whole blood sample in the ratio of approximately 50-1000:1reagent to blood. The sample and reagent can be rapidly mixed to achieveerythrolysis and conversion of hemoglobin to the chromogen. The sampleand reagent mixture may then be presented to an absorbancespectrophotometer where the optical density of the chromogen formed ismeasured. When the ligand is imidazole the measurement can be madebetween 540 nm and 550 nm. The total hemoglobin concentration in thesample is related to the optical density of the converted chromogen.

The invention will now be described with reference to examples which areintended to illustrate and not limit the scope of the claimed invention.

EXAMPLE 1

0.4 Moles of imidazole (27.2 g) (Sigma Chemical Company) and 0.1 mole ofsodium hydroxide (4.0 g) (Fisher Chemical Company) were added toapproximately 900 ml of distilled water in a graduated cylinder. Thereagents were dissolved using a magnetic stirrer. 3.3 mL of lauryldimethylamine oxide (nonionic surfactant, 30% solution, Stepan ChemicalCompany, Northfield, Ill.) was added with stirring and the volumeadjusted to 1000 ml by addition of distilled water. The solution wasfiltered through a 0.2 micron Nalgene filter and stored at roomtemperature until used. This formulation is subsequently referred to asImidazole 0.4.

Other reagents were prepared in the same manner except that the amountof imidazole was varied between 0.1 mole (6.8 g) and 1.0 mole (68.1 g).These formulations are subsequently referred to as Imidazole "X" where Xrefers to the molar concentration.

EXAMPLE 2

Twelve (12) microliters of whole blood was mixed with 3.0 mL ofImidazole 0.4. The absorption spectrum was recorded from 780 nm to 480nm with a Hewlett Packard 8452A Diode Array Spectrophotometer ("HP").FIG. 1 shows a peak at 544 nm with a shoulder around 580 nm. Forcomparison, the spectra of oxy and de-oxy hemoglobin at the sameconcentration are included.

EXAMPLE 3

Whole blood (12) microliters was rapidly mixed with 3.0 ml of Imidazolereagents ranging from 0.1 to 1.0M. The absorbance was monitored at 544nm for 60 seconds. The change in absorbance between 10 seconds post mixand 60 seconds post mix was measured and tabulated as

    ______________________________________                                        Imidazole Conc. Drift (mA)                                                    ______________________________________                                        0.1             -80                                                           0.25            -10                                                           0.4             0                                                             0.66            0                                                             1.0             0                                                             ______________________________________                                    

EXAMPLE 4

A series of formulations with varying NaOH, surfactant and imidazoleconcentrations were made and tested for completeness of lysis andstability of signal. Twelve (12) microliters of whole blood were mixedwith reagent. Absorbance at 700 nm was observed for completeness oflysis and at 544 nm for completeness of the conversion to chromogen.Completeness of lysis is indicated by a "+"; presence of drift isindicated by a "+"; "++" indicates pronounced drift.

Although it is desirable to have the reaction complete, that is completelysis and lack of drift, within a short period of time, it is possibleto use a drifting signal if the timing of reagent addition andabsorbance measurement is controlled.

    ______________________________________                                        NaOH (M) Ammonix LO (%)                                                                              Imidazole  Lysis Drift                                 ______________________________________                                        0.01     0.01          0.1        +     +                                     0.01     0.01          1.0        -     ++                                    0.01     0.1           0.1        +     +                                     0.01     0.1           1.0        +     -                                     0.1      0.01          0.1        +     +                                     0.1      0.1           1.0        +     -                                     0.1      0.01          0.1        +     +                                     0.1      0.1           1.0        +     -                                     ______________________________________                                    

EXAMPLE 5

For this and Examples 6 and 7, Imidazole 0.4 was used exclusively.Manipulated blood samples were prepared to give a range of hematocritvalues. The samples were prepared by centrifuging an EDTA anticoagulatedblood sample at 500 g for 10 minutes. The plasma was added to a normalblood sample to prepare a reduced hematocrit sample. The buffy coat wasreserved to give an elevated white blood cell count sample with a normalhematocrit. The sedimented red blood cells were analyzed as an elevatedhematocrit sample. Hematocrits and white blood cell counts weredetermined on a Baker 4000 analyzer. The absorbance of the samplediluted 1 to 1:251 with Imidazole 0.4 was read at 540 nm on aHewlett-Packard 8452A Diode Array Spectrophotometer. The results areshown below.

    ______________________________________                                        Sample   Hematocrit WBC Count   Absorbance (mA)                               ______________________________________                                        Low      10.6       0.7         105.6                                         High WBC 45.9       19.1        421.0                                         Normal   42.0       7.3         397.5                                         High     74.1       8.0         722.5                                         ______________________________________                                    

EXAMPLE 6

A kit for the determination of hemoglobin by the method of Drabkin wasused to compare the performance of the Imidazole 0.4 reagent with anestablished reference method. The kit, Catalog No. 525-A, was obtainedfrom Sigma Diagnostics, P.O. Box 14508, St. Louis, Mo., 63178, and wasreconstituted and used according to the instructions.

A normal sample of whole blood was manipulated to give hemoglobinconcentrations from zero to approximately 20 g/dL. Samples were analyzedby the Drabkin's method and using Imidazole 0.4. The Drabkin's samplewas reacted at a 1 to 251 ratio and the Imidazole 0.4 at a ratio of 1 to101. All reaction mixtures were read at 40 nm in a Hewlett Packard DiodeArray Spectrophotometer. Although the Imidazole 0.4 reaction is completewithin 10 seconds, the reaction mixtures were allowed to incubate forapproximately 30 minutes before reading for convenience.

    ______________________________________                                        Sample   Hb by Drabkin's                                                                            Imidazole A540 (mA)                                     ______________________________________                                        1        0            0.6                                                     2        2.8          204.4                                                   3        7.1          511.5                                                   4        9.9          735.4                                                   5        13.9         983.7                                                   6        15.2         1148.6                                                  7        15.9         1182.0                                                  8        17.2         1242.1                                                  9        17.6         1348.8                                                  10       19.8         1435.3                                                  11       19.9         1545.0                                                  ______________________________________                                    

The correlation coefficient of the Imidazole 0.4 reagent with theDrabkin's reagent is r=+0.9976, the Slope is 75.2 mA/g/dL, and theintercept is -12.0 mA. The excellent linear correlation of the A540given by the Imidazole 0.4 reagent with the standard Drabkin's reagentdemonstrates the equivalence of results by the two methods and allowsthe use of the non-poisonous formulation of the current inventioninstead of the cyanide containing standard method.

EXAMPLE 7

The Imidazole 0.4 reagent was compared to a commercially available,semi-automated hematology analyzer, the Coulter JT, using Lysis SIII(which contains potassium cyanide) and Isoton III reagents, [all fromCoulter Diagnostics, Hialeah, Fla.] A series of manipulated whole bloodsamples were prepared as above with hemoglobin concentrations rangingfrom 0 to about 25 g/dL. The samples were analyzed on the Coulter JTanalyzer as described in the instrument's manual. The Imidazole 0.4reagent was reacted with samples at a ratio of 1 to 126 by dispensing 40microliters blood sample using a positive displacement pipette(Absoluter Pipettor, Tri-Continent Scientific Company, Grass Valley,Calif.) into a tube and adding 5.0 ml of the Imidazole 0.4 reagent withan Eppendorf Repipettor (Brinkman Instruments, Inc., Westbury, N.Y.) Theabsorbance at 540 nm of the reacted samples was read on a Beckman DU-7Spectrophotometer (Beckman Instruments, Fullerton, Calif.) equipped witha sipper. Determinations with the Imidazole 0.4 were made in duplicateand the results averaged.

The strong linear correlation (correlation coefficient, r=0.9998; Slope,m=49.94 mA/g/dL, intercept b=4.4 mA) demonstrates that the currentinvention gives results strictly comparable to the known commercialinstrument.

    ______________________________________                                        Sample    Coulter (g/dL)                                                                            Imidazole A540 (mA)                                     ______________________________________                                        1         0.1         -0.4                                                    2         5.5         283.6                                                   3         8.0         406.6                                                   4         10.6        535.2                                                   5         12.8        645.7                                                   6         14.3        732.5                                                   7         18.7        935.2                                                   8         19.8        983.6                                                   9         20.3        1016.2                                                  10        26.0        1303.4                                                  ______________________________________                                    

EXAMPLE 8

Fifty-two clinical samples, whose hemoglobin concentration ranged from12 g/dL to 17.5 g/dL, were analyzed on a calibrated Cell-Dyn 3500system, a fully automated hematology analyzer currently sold by AbbottLaboratories, Diagnostics Division, Santa Clara, Calif., and also on afully automated, experimental hematology analyzer under developmentalong with the current invention (However, any automated hematologyanalyzer could easily be adapted to accept the reagent and method ofthis invention.). The Cell-Dyn 3500 hemoglobin reagent containspotassium cyanide and the reactant of blood hemoglobin iscyanmethemoglobin. The whole blood dilution factor for hemoglobinmeasurement is 1:301 and the detection wavelength of the reactant is 540nm. The experimental hemoglobin reagent contains 0.4 moles of imidazole,0.1 mole of sodium hydroxide, and 0.1% (w/v) lauryl dimethylamine oxidein deionized water. The pH of the reagent was 13.0 without adjustment.The whole blood dilution factor for the CD 3500 hemoglobin measurementin the system is 1:125 and the reaction time is 10 seconds. Thedetection wavelength of the converted chromogen is 540 nm. Thehemoglobin results of the experimental analyzer were then compared tothat of the Cell-Dyn 3500 by linear regression analysis.

As can be seen in the regression statistics in FIG. 2, the performanceof the two methods is essentially equivalent. Correlationcoefficient=0.99; Slope=0.97; and Y-Intercept=0.34.

The strong linear correlation demonstrates that the current inventiongives results strictly comparable to the known commercial instrument.

EXAMPLE 9

Fifty-two clinical samples, whose hemoglobin concentration ranged from12 g/dL to 17.5 g/dL, were analyzed on a calibrated Coulter STKS system,a fully automated hematology analyzer in the market, and also on acalibrated experimental hematology analyzer under development with thecurrent invention (as in EXAMPLE 8). Coulter STKS hemoglobin reagentcontains potassium cyanide and the reactant of blood hemoglobin iscyanmethemoglobin. The detection wavelength of the reactant is 540 nm.The experimental hemoglobin reagent contains 0.4 moles of imidazole, 0.1mole of sodium hydroxide, and 0.1% (w/v) lauryl dimethylamine oxide. ThepH of the reagent was 13.0 without adjustment. The whole blood dilutionfactor for hemoglobin in the system is 1:125 and the reaction time is 10seconds. The detection wavelength of the converted chromogen is 540 nm.The hemoglobin results of the experimental analyzer were then comparedto that of the Coulter STKS by linear regression analysis. As can beseen in the regression statistics in FIG. 3, the performance of the twomethods is essentially equivalent. Correlation coefficient=0.98;Slope=1.02; and Y-Intercept=-0.21.

The strong linear correlation demonstrates that the current inventiongives results strictly comparable to that of the cyan met hemoglobinmethod of the well known commercial hematology instrument.

EXAMPLE 10

Twelve clinical samples whose hemoglobin concentration ranged from 7.0to 17.6 g/dL were analyzed on a calibrated experimental analyzer system(as in EXAMPLE 8) using a freshly prepared hemoglobin reagent containing0.4 moles of imidazole, 0.1 mole of sodium hydroxide and 0.1% (w/v) oflauryl dimethylamine oxide in deionized water. The pH of the reagent was13.0 without adjustment. The same twelve samples were rerun, withoutre-calibration, with a hemoglobin reagent of the same composition butaged at ambient temperature for six months to evaluate the stability ofthe reagent.

The results of the control reagent was compared to that of the sixmonths old test reagent and the linear regression data are presented inFIG. 4. No significant difference was found between the two runs,indicating that the reagent is stable at least six months at ambienttemperature. Correlation coefficient=0.998; Slope=1.01; andY-Intercept=-0.27.

EXAMPLE 11

To check the stability of the reagent of the current inventioncontaining 0.4 moles of imidazole, 0.1 mole of sodium hydroxide and 0.1%(w/v) of lauryl dimethylamine oxide in deionized water, the conversiontime and the spectrum of the converted chromogen in a less than onemonth old reagent was compared to that of the reagent stored for threemonths at 45° C. For this study, twenty four (24) microliters of a freshnormal blood was placed in a cuvette and 3.0 ml of the appropriatereagent was rapidly added from a serological pipette. Then, the reactionmixture was mixed with two aspiration/dispense cycles with a Pasteurpipette.

The results are presented in FIG. 5. As evidenced by the trace, mixingwas complete within 10 seconds. The reaction was followed at 544(Absorption max.), 580 (shoulder), 500 (valley), and 700 (turbiditycheck) nm (top to bottom traces). The 700 nm reading indicatescompleteness of lysis. Both control and 45° C. stored reagents appear tohave identical kinetics of reaction. The conversion time was less than10 seconds. Arrehnius' extrapolation from these data indicate that thereagent formulation of the current invention is stable for over a yearat ambient temperature.

EXAMPLE 12

A normal blood sample with hemoglobin concentration of 15.8 g/dL, asdetermined on a calibrated Cell-Dyn 3500 system, was used to evaluatethe level of turbidity interference in the hemoglobin measurement from alipemic sample in the current invention. This is a known problem inhemoglobin measurement by many methods including ICSH cyanmethemoglobinmethod. Lipid-Trol (PN# L2648) was obtained from Sigma Chemical Company.The product contained 1330 mg/dl triglyceride and 840 mg/dL cholesterol.Five dilution's of the product were made with isotonic saline to thefinal concentrations of triglyceride and cholesterol indicated in theTable below to sufficiently cover the clinical range. Twenty (20)microliters of these solutions (20 microliters of saline was added tothe control preparation) were added to the 1:125 dilution of the bloodsample with the hemoglobin reagent of the current invention as inEXAMPLE 10, mixed and the optical densities of each sample was measuredusing a 1 cm cuvette at 540 nm (for hemoglobin) and 700 nm (forturbidity) in a Turner Model 690 spectrophotometer. The concentration ofhemoglobin in each preparation was calculated using the hemoglobin valueof the control (Cell-Dyn 3500 system hemoglobin value on the same bloodsample).

Note that the O.D. of the converted chromogen of the current inventionhas a linear relationship to the concentration of hemoglobin.

    ______________________________________                                        TRIG/CHOL 540 nm O.D.                                                                              700 nm O.D.                                                                              Hb Conc. (g/dL)                               ______________________________________                                        1330/840  0.82       0.03       15.6                                          1064/672  0.82       0.03       15.6                                          798/504   0.82       0.03       15.6                                          532/336   0.84       0.03       15.9                                          266/168   0.82       0.03       15.6                                          0/0 (control)                                                                           0.83       0.03       15.8                                          ______________________________________                                    

As the data show, no significant difference either in the turbidity (700nm O.D.) measurement or in the hemoglobin value (540 nm O.D.) wasobserved due to the addition of triglyceride and cholesterol in thesesample preparations.

EXAMPLE 13

A normal blood sample with hemoglobin concentration of 15.6 g/dL, asdetermined on a calibrated Cell-Dyn 3500 system, was used to evaluatethe level of bilirubin interference in the hemoglobin measurement from ahyperbilirubinemia patient's sample in the current invention. This is aknown problem in hemoglobin measurement by many methods including NCCLScyanmethemoglobin method. A Bilirubin Standard (Catalog No. 550-11) wasobtained from Sigma Chemical Company. The product contained 30 mg/dLbilirubin when the total content in the vial was reconstituted with 1.5ml of de ionized water. Five dilution's of the product was made withisotonic saline to the final concentrations of bilirubin indicated inthe Table below to sufficiently cover the clinical range. Twenty (20)microliters of these solutions (20 microliters of saline was added tothe control preparation) were added to the 1:125 dilution's of the bloodsample with the hemoglobin reagent of the current invention as inEXAMPLE 10, mixed and the optical densities of each sample was measuredusing a 1 cm cuvette at 540 nm (for hemoglobin) and 700 nm (forturbidity) in a Turner Model 690 spectrophotometer. The concentration ofhemoglobin in each preparation was calculated using the hemoglobin valueof the control (Cell-Dyn 3500 system hemoglobin value on the same bloodsample). Note that the O.D. of the converted chromogen of the currentinvention has a linear relationship to the concentration of hemoglobin.

    ______________________________________                                        Bilirubin                                                                     in mg/dL 540 nm O.D.                                                                              700 nm O.D.                                                                              Hb Conc. in gm/dL                              ______________________________________                                        0.0 (control)                                                                          0.81       0.023      15.6                                            4.0     0.81       0.025      15.5                                           10.0     0.81       0.027      15.6                                           15.0     0.81       0.025      15.6                                           20.0     0.82       0.024      15.7                                           30.0     0.83       0.031      15.9                                           ______________________________________                                    

As the data show, no significant increase either in the turbidity (700nm O.D.) measurement or in the hemoglobin value (540 nm O.D.) wasobserved due to the addition of bilirubin in these sample preparations.The results indicate that hyperbilirubinemia patients' samples will notinterfere with the current invention in the determination of total bloodhemoglobin concentration.

While certain representative embodiments and details have been shown forpurpose of illustrating the invention, various changes and modificationscan be made therein without departing from the scope of the inventiondefined in the claims.

What is claimed is:
 1. A cyanide-free reagent for use in totalhemoglobin measurement comprising an aqueous solution of:(a) acyanide-free ligand selected from the group consisting of imidazole,imidazole derivatives, N-hydroxyacetamide, N-hydroxyl amine, pyridine,oxazole, thiazole, pyrazole, pyrimidine, purine, quinoline, andisoquinoline and the ligand is present at a concentration of 0.1 to 2.0Molar; and (b) a surfactant selected from the group consisting of lauryldimethylamine oxide and octylphenoxy polyethoxy ethanol present at aconcentration from about 0.1% to about 1.0% (w/v),wherein the pH of thereagent is adjusted to between 11 and 14, and wherein the ligandsubstantially completely ligates heme molecules of a blood sample towhich the reagent has been added.
 2. The reagent of claim 1 wherein thesurfactant is lauryl dimethylamine oxide.
 3. The reagent of claim 2wherein the lauryl dimethylamine oxide is present at a concentration ofabout 0.1% (w/v).
 4. The reagent of claim 1 wherein the ligand isimidazole.
 5. The reagent of claim 1 wherein the imidazole is present ata concentration of about 0.4M.
 6. The reagent of claim 1 wherein theligand is imidazole and the surfactant is lauryl dimethylamine oxide. 7.The reagent of claim 1 wherein the reagent pH is adjusted using amonovalent base.